Peptidyl prolyl cis-trans isomerases

ABSTRACT

The present invention provides a method for producing a secretable polypeptide in a host cell. In the method, a peptidyl prolyl cis-trans isomerase is overexpressed in a host cell, thereby increasing the yield of the secreted polypeptide.

The present invention relates to a novel enzyme. In particular, theinvention relates to a novel cyclophilin-like peptidyl prolyl cis-transisomerase.

In most protein over-production strategies, strong promoters capable ofdirecting very high levels of transcription are used to over-expressgenes encoding heterologous and homologous proteins. Limitations inprotein secretion are likely to be due to bottlenecks at thetranslational and post-translational levels (Tsuchiya, K. et al., (1992)Applied Microbiology and Biotechnology 38:109-114). Proper folding ofthe protein is required for export competence. Overexpressedheterologous proteins may fold improperly and then be degraded duringprotein traffic through the secretory pathway. Therefore, the correctionof folding defects is desirable in order to increase protein secretion.

The folding of a protein is catalysed by an number of factors, includingtwo isomerase families, namely protein disulphide isomerase, catalysingdisulphide bond formation, and peptidyl prolyl cis-trans isomerase,catalysing the isomerisation of Xaa-Proline bonds.

Overexpression of protein disulphide isomerase in S. cerevisiae resultsin an increase in secretion of human platelet-derived growth factor(Robinson et al., 1994). However, the overproduction of A. niger proteindisulphide isomerase did not result in an increase in secretion of henegg white lysozyme (HEWL) or glucoamylase (Ngiam C., Jeenes, D. J. J.,Punt, P. J., van den Hondel, C. A. M. J. J., Archer, D. A. (1998);Characterisation of a foldase, PDIA, in the protein secretory pathway ofAspergillus niger; submitted.).

One of the slowest steps in protein folding is the cis-transisomerisation of Xaa-proline bonds. This isomerisation is markedlyaccelerated when peptidyl prolyl cis-trans isomerases are present.Peptidyl prolyl cis-trans isomerases of the cyclophilin family fromdifferent organisms have been shown to possess foldase activity in vitro(Schönbrunner E. R., Mayer S., Tropschug M., Fischer G., Takahashi N.,Schmid, F. (1991); Catalysis of protein folding by cyclophilins fromdifferent species. J Biol Chem 266: 3630-3635). These isomerases areinhibited by the immuno-suppressant drug cyclosporin A. The effects onsecretion of heterologous peptides by overexpression of peptidyl prolylcis-trans isomerases are however not known in the prior art.

Proteins active in the E.R. are targeted to this compartment by acarboxy terminal extension of 4 amino acids. In A. niger HDEL (SEQ IDNO: 7) and KDEL (SEQ ID NO: 9) have been reported to function as an E.R.retention signal (Jeenes D. J., et al., (1997) Gene 193:151-156).

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a methodfor producing a secretable polypeptide in an host cell, comprisingoverexpressing a peptidyl prolyl cis-trans isomerase in the cell,thereby increasing the yield of the secreted polypeptide.

In a second aspect, the invention relates to a polypeptide possessingfoldase activity characterised by having a capability to catalyse thecis-trans isomerisation of a peptide bond on the N terminal side ofproline residues in polypeptides, having a signal sequence at theN-terminus and an endoplasmic reticulum retention signal at theC-terminus, and a molecular weight of 20.7 kDa and a deduced isoelectricpoint of 6.27.

In a third aspect, the invention relates to a polypeptide possessingfoldase activity characterised by having a capability to catalyse thecis-trans isomerisation of a peptide bond on the N terminal side ofproline residues in polypeptides, encoded by a nucleic acid capable ofhybridising under conditions of low, medium or high stringency with a 17base oligonucleotide derived from SEQ ID No. 1.

In a fourth aspect, the invention relates to a polypeptide possessingfoldase activity characterised by having a capability to catalyse thecis-trans isomerisation of a peptide bond on the N terminal side ofproline residues in polypeptides, encoded by a nucleic acid capable ofhybridising under conditions of low, medium or high stringency with a 20base oligonucleotide derived from SEQ ID No. 2.

In a fifth aspect, the invention relates to a polypeptide possessingfoldase activity characterised by having a capability to catalyse thecis-trans isomerisation of a peptide bond on the N terminal side ofproline residues in polypeptides, which is at least 40% homologous toSEQ. ID. No. 2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of plasmid pPD23, which encodes A. nigercypB.

FIG. 2 shows an alignment of the cypB gene (SEQ ID NO: 5); with peptidylcis-trans isomerases from Orpinimyces, M. musculus and H. sapiens (SEQID NOS: 10-12, respectively, in order of appearance).

FIG. 3 shows an optimised alignment between the cypB gene (SEQ ID NO: 5)and the Orpinimyces PPI gene (SEQ ID NO: 10).

FIG. 4 is a representation of pLIP4, which encodes the lipA gene.

FIG. 5 is a representation of ppd23d13.

FIG. 6 is a representation of ppd23d14.

FIG. 7 is a representation of ppd38d3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the overexpression of a peptidyl-prolylcis-trans isomerase polypeptide (PPI) to increase the expression ofsecreted polypeptides from host cells. It has been found that increasinglevels of PPI in cells is effective to facilitate secretion ofpolypeptides from the cell. PPI is believed to prevent the retention ofpolypeptide products in the ER, and their subsequent degradation, byreducing the level of misfolding thereof. Thus, the invention isparticularly suitable for increasing the yield of secreted polypeptidesfrom cells.

As used herein, the term “peptidyl-prolyl cis-trans isomerase”polypeptide (PPI) is used to denote an enzyme which is capable ofcatalysing the cis-trans isomerisation of a peptide bond on the Nterminal side of proline residues in polypeptides. PPIs are ubiquitous,and several examples are known in the art. Examples include cyclophilin(see, for example, Bergsma et al. (1991) J. Biol. Chem.266:23204-23214), parvulin, SurA (Rouviere and Gross, (1996) Genes Dev.10:3170-3182) and FK506 binding proteins FKBP51 and FKBP52. PPI isresponsible for the cis-trans isomerisation of peptidyl-prolyl bonds inpolypeptides, thus promoting correct folding. The invention includes anypolypeptide having PPI activity. This includes chaperone polypeptides,or fragments thereof, which may possess PPI activity (Wang & Tsou,(1998) FEBS lett. 425:382-384). Preferably, the invention relates to PPIpolypeptides of the cyclophilin family.

Advantageously, the host cells are transformed host cells which expressa heterologous gene product. Particularly in cases where theheterologous gene product is overexpressed, the tendency for theresulting polypeptides to be misfolded, and thus degraded in the ER asset forth above, is increased. Under these circumstances, therefore,overexpression of PPI in accordance with the invention is highlyadvantageous.

However, the invention may also be used to increase the production ofhomologous polypeptides in host cells. For example, the invention isuseful where transcription of homologous polypeptides is increased, as aresult of an increase in cell activity, caused by natural biologicalprocesses or by administration of agents capable of up regulating genetranscription. Moreover, cells may be transformed with expressionsystems capable of a causing upregulation of the endogenous genes, forexample expression systems encoding transcription factors which areactive on endogenous promoters.

Similarly, upregulation of PPI expression may be achieved by increasingthe expression of endogenous PPI or by transforming the host cell with acoding sequence capable of producing PPI at elevated levels.Advantageously, host cells are transformed with a PPI-encoding sequence.

In a preferred embodiment, therefore, the invention relates to a methodfor producing a secretable polypeptide in a host cell, comprisingcotransfecting the cell with a first coding sequence encoding thepolypeptide and a second coding sequence encoding a peptidyl prolylcis-trans isomerase.

Advantageously, the invention relates to a method for expressing asecretable polypeptide in a host cell, comprising the steps of:

a) transforming the cell with a coding sequence expressing a peptidylprolyl cis-trans isomerase according to the invention;

b) transforming the cell with a coding sequence expressing a desiredpolypeptide; and

c) culturing the cell to produce the polypeptide.

As used herein, transfection and transformation are consideredequivalent, and include any form of insertion of DNA into cells,including viral transduction, electroporation and conventionaltransfection techniques. The coding sequences encoding peptidyl prolylcis-trans isomerase and the desired polypeptide may be inserted into thecells on vectors, or independently as naked DNA. The use of vectors ispreferred. Where the peptidyl prolyl cis-trans isomerase and the desiredpolypeptide are present on separate vectors, either one of the separatevectors may be inserted into the host cell before the other. The orderof insertion is not important, as long as increased levels of peptidylprolyl cis-trans isomerase are obtained in the host cell during theexpression of the desired polypeptide.

Advantageously, the peptidyl prolyl cis-trans isomerase and the desiredpolypeptide may be present on the same vector.

Preferably, host cells may be constructed wherein a coding sequenceexpressing peptidyl prolyl cis-trans isomerase is integrated into thehost cell and genome. This can be achieved using an integratingexpression vector to transform the cell with the said coding sequence.

As noted above, the coding sequence expressing, the peptidyl prolylcis-trans isomerase is preferably incorporated into a suitable vector.As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous DNA into cells for either expression orreplication thereof. Selection and use of such vehicles are well withinthe skill of the artisan. Many vectors are available, and selection ofappropriate vector will depend on the intended use of the vector and thehost cell to be transformed with the vector. Each vector containsvarious components depending on its function and the host cell for whichit is compatible. The vector components generally include, but are notlimited to, one or more of the following: an origin of replication, oneor more marker genes, an enhancer element, a promoter, a transcriptiontermination sequence and a signal sequence.

Most expression vectors are shuttle vectors, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another class of organisms for expression. For example, a vectormay be cloned in E. coli and then the same vector is transfected intoyeast or other fungal cells even though it is not capable of replicatingindependently of the host cell chromosome. DNA can also be amplified,for example by PCR, and be directly transfected into the host cellswithout any replication component.

Advantageously, an expression vector may contain a selection gene, alsoreferred to as selectable marker. This gene encodes a protein necessaryfor the survival or growth of transformed host cells grown in aselective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that confer resistance toantibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate ortetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available from complex media.

As to a selective gene marker appropriate for yeast and other fungalorganisms, any marker gene can be used which facilitates the selectionfor transformants due to the phenotypic expression of the marker gene.Suitable markers for yeast are, for example, those conferring resistanceto antibiotics G418, hygromycin or bleomycin, or provide for prototrophyin an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1,or HIS3 gene.

Since the replication of vectors is conveniently done in E. coli, an E.coli genetic marker and an E. coli origin of replication areadvantageously included. These can be obtained from E. coli plasmids,such as pBR322, Bluescript© vector or a pUC plasmid, e.g. pUC18 orpUC19, which contain both E. coli replication origin and E. coli geneticmarker conferring resistance to antibiotics, such as ampicillin.

Expression and cloning vectors usually contain a promoter that isrecognised by the host organism and is operably linked to nucleic acidencoding peptidyl prolyl cis-trans isomerase. Such a promoter may beinducible or constitutive. The promoters are operably linked to DNAencoding the peptidyl prolyl cis-trans isomerase by removing thepromoter from the source DNA by restriction enzyme digestion andinserting the isolated promoter sequence into the vector. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the peptidyl prolyl cis-transisomerase coding sequence. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A control sequence“operably linked” to a coding sequence is ligated in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequences.

Suitable promoting sequences for use with yeast hosts may be regulatedor constitutive and are preferably derived from a highly expressedfungal gene. Fungal promoters are known in the literature (for example,see Gurr, et al., (1987) The structure and organisation of nuclear genesof filamentous fungi. In Kinghorn, J. R. (ed), Gene Structure inEukaryotic Microbes, IRL Press, Oxford, pp. 93-139). Yeast promoters mayalso be used, such as the promoter of the yeast TRP1 gene, the ADHI orADHII gene, the acid phosphatase (PH05) gene, a promoter of the yeastmating pheromone genes coding for the a- or α-factor or a promoterderived from a gene encoding a glycolytic enzyme such as the promoter ofthe enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase,phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglyceratemutase, pyruvate kinase, triose phosphate isomerase, phosphoglucoseisomerase or glucokinase genes, the S. cerevisiae GAL 4 gene, the S.pombe nmt 1 gene or a promoter from the TATA binding protein (TBP) gene.Furthermore, it is possible to use hybrid promoters comprising upstreamactivation sequences (UAS) of one yeast gene and downstream promoterelements including a functional TATA box of another yeast gene, forexample a hybrid promoter including the UAS(s) of the yeast PH05 geneand downstream promoter elements including a functional TATA box of theyeast GAP gene (PH05-GAP hybrid promoter). A suitable constitutive PHO5promoter is e.g. a shortened acid phosphatase PH05 promoter devoid ofthe upstream regulatory elements (UAS) such as the PH05 (−173) promoterelement starting at nucleotide −173 and ending at nucleotide −9 of thePH05 gene.

In connection with the present invention, the use of fungal organisms,such as a filamentous fungi, for example those used in the biotechnologyindustry; preferably Aspergillus, Trichoderma, Neurospora, Mucor orPenicillium, is preferred. More specifically, preferred host organismsinclude A. nidulans, A. tubigensis, A. sojae, A. awamori, A. oryzae, A.japonicus, A. aculeatus, N. crassa, T. reesei and T. viride. A preferredhost organism for the expression of the nucleic acid constructs of thepresent invention and/or for the preparation of the heterologouspolypeptides according to the present invention is an organism of thegenus Aspergillus, advantageously Aspergillus niger. In this regard, atransgenic Aspergillus according to the present invention can beprepared by following the teachings of Rambosek, J. and Leach, J. 1987(Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit.Rev. Biotechnol. 6:357-393), Davis R. W. 1994 (Heterologous geneexpression and protein secretion in Aspergillus. In: Martinelli S. D.,Kinghorn J. R. (Editors) Aspergillus: 50 years on. Progress inindustrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560),Ballance, D. J. 1991 (Transformation systems for Filamentous Fungi andan Overview of Fungal Gene structure. In: Leong, S. A., Berka R. M.(Editors) Molecular Industrial Mycology. Systems and Applications forFilamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1-29) and TurnerG. 1994 (Vectors for genetic manipulation. In: Martinelli S. D.,Kinghorn J. R. (Editors) Aspergillus: 50 years on. Progress inindustrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666).The following commentary provides a summary of those teachings forproducing transgenic Aspergillus according to the present invention.

In order to prepare the transgenic Aspergillus, expression constructsare prepared by inserting a heterologous nucleotide sequence (such as anucleotide sequence coding for an amylase enzyme) into a constructdesigned for expression in filamentous fungi.

Several types of constructs used for heterologous expression have beendeveloped. The constructs contain the promoter according to the presentinvention which is active in fungi. The heterologous nucleotide sequencecan be fused to a signal sequence which directs the protein encoded bythe heterologous nucleotide sequence to be secreted. Usually a signalsequence of fungal origin is used. A terminator active in fungi may alsobe employed.

Another type of expression system has been developed in fungi where theheterologous nucleotide sequence is fused to a fungal gene encoding astable protein. This can stabilise the protein encoded by theheterologous nucleotide sequence which encodes a desired polypeptide. Insuch a system a cleavage site, recognised by a specific protease, can beintroduced between the fungal protein and the protein encoded by theheterologous nucleotide sequence, so the produced fusion protein can becleaved at this position by the specific protease thus liberating theprotein encoded by the heterologous nucleotide sequence. By way ofexample, one can introduce a site which is recognised by a KEX-2 likepeptidase found in at least some Aspergilli (Broekhuijsen et al 1993 JBiotechnol 31 135-145). Such a fusion leads to cleavage in vivoresulting in protection of the expressed product and not a larger fusionprotein.

Heterologous expression in Aspergillus has been reported for severalgenes coding for bacterial, fungal, vertebrate and plant proteins. Withregard to product stability and host strain modifications, someheterologous proteins are not very stable when they are secreted intothe culture fluid of fungi. Most fungi produce several extracellularproteases which degrade heterologous proteins. To avoid this problemspecial fungal strains with reduced protease production have been usedas host for heterologous production.

For the transformation of filamentous fungi, several transformationprotocols have been developed for many filamentous fungi (Ballance 1991,ibid). Many of them are based on preparation of protoplasts andintroduction of DNA into the protoplasts using PEG and Ca²⁺ ions. Thetransformed protoplasts then regenerate and the transformed fungi areselected using various selective markers. Among the markers used fortransformation are a number of auxotrophic markers such as argB, trpC,niaD and pyrG, antibiotic resistance markers such as benomyl resistance,hygromycin resistance and phleomycin resistance. A commonly usedtransformation marker is the amdS gene of A. nidulans which in high copynumber allows the fungus to grow with acrylamide as the sole nitrogensource.

Transcription of a DNA encoding peptidyl prolyl cis-trans isomerase byfungal organisms may be increased by inserting an enhancer sequence intothe vector. Enhancers are relatively orientation and positionindependent.

An expression vector includes any vector capable of expressing peptidylprolyl cis-trans isomerase encoding nucleic acids that are operativelylinked with regulatory sequences, such as promoter regions, that arecapable of expression of such DNAs. Thus, an expression vector refers toa recombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector, that upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those with ordinaryskill in the art and include those that are replicable in eukaryoticand/or prokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

Construction of vectors according to the invention employs conventionalligation techniques. Isolated plasmids or DNA fragments are cleaved,tailored, and religated in the form desired to generate the plasmidsrequired. If desired, analysis to confirm correct sequences in theconstructed plasmids is performed in a known fashion. Suitable methodsfor constructing expression vectors, preparing in vitro transcripts,introducing DNA into host cells, and performing analyses for assessingpeptidyl prolyl cis-trans isomerase expression and function are known tothose skilled in the art. Gene presence, amplification and/or expressionmay be measured in a sample directly, for example, by conventionalSouthern blotting, Northern blotting to quantitate the transcription ofmRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation,using an appropriately labelled probe which may be based on a sequenceprovided herein. Those skilled in the art will readily envisage howthese methods may be modified, if desired.

The same or similar considerations will apply to the design of a vectorencoding the desired polypeptide. Although it is not necessary, it ispossible for the PPI and the desired polypeptide to be encoded on thesame vector, whether episomal or integrating, and be expressedtherefrom. Preferably, the desired polypeptide is a polypeptide encodedby a heterologous nucleotide sequence not derived from the hostorganism.

Typical examples of a nucleotide sequence encoding a desired polypeptideinclude sequences coding for proteins and enzymes that modify metabolicand catabolic processes. The heterologous nucleotide sequence may codefor an agent for introducing or increasing pathogen resistance. Theheterologous nucleotide sequence may code for a non-native protein of afilamentous fungus, preferably of the genus Aspergillus, or a compoundthat is of benefit to animals or humans. Examples of nucleotidesequences according to the invention include pectinases, pectindepolymerases, polygalacturonases, pectate lyases, pectin lyases, hexoseoxidase, oxidoreductases, lipases, glucan lyase, rhamnogalacturonases,hemicellulases, endo-β-glucanases, arabinases, or acetyl esterases, orcombinations thereof, as well as antisense sequences thereof. Thedesired polypeptide may be a protein giving nutritional value to a foodor crop. Typical examples include plant proteins that can inhibit theformation of anti-nutritive factors and plant proteins that have a moredesirable amino acid composition (e.g. a higher lysine content than anon-transgenic plant).

The desired polypeptide may be an enzyme that can be used in foodprocessing such as chymosin, thaumatin and α-galactosidase. The desiredpolypeptide may moreover be any one of a pest toxin, ADP-glucosepyrophosphorylase (e.g. see EP-A-0455316), a glucanase or genomicβ-1,4-endoglucanase.

The heterologous nucleotide sequence may code for an intron of aparticular nucleotide sequence, wherein the intron can be in sense orantisense orientation.

The heterologous nucleotide sequence can be the nucleotide sequencecoding for the arabinofuranosidase enzyme which is the subject of PCTpatent application PCT/EP96/01009 (incorporated herein by reference).The heterologous nucleotide sequence can be any of the nucleotidesequences coding for the ADP-glucose pyrophosphorylase enzymes which arethe subject of PCT patent application PCT/EP94/01082 (incorporatedherein by reference). The heterologous nucleotide sequence can be any ofthe nucleotide sequences coding for the α-glucan lyase enzyme which aredescribed in PCT patent application PCT/EP94/03397 (incorporated hereinby reference). The heterologous nucleotide sequence can be any of thesequences coding for T. languinosus amylase, as described in PCT patentapplication PCT/EP95/02607, incorporated herein by reference. Theheterologous nucleotide sequence can be any of the nucleotide sequencescoding for the glucanase enzyme which are described in PCT patentapplication PCT/EP96/01008 (incorporated herein by reference).

In a preferred aspect of the invention, the nucleic acid encoding PPIwill also include, operatively linked thereto, an ER retention signal.Preferably, the ER retention signal is a tetrapeptide, which isadvantageously HDEL (SEQ ID NO: 7), HEEL (SEQ ID NO: 8) or KDEL (SEQ IDNO: 9). The ER retention signal targets the polypeptide to the ER andcauses it to be retained therein.

According to the second aspect of the present invention, there isprovided a polypeptide which possesses a foldase activity and ischaracterised by having a capability to catalyse the cis-transisomerisation of a peptide bond on the N terminal side of prolineresidues in polypeptides, having a signal sequence at the N terminus andan endoplasmic reticulum retention signal at the C terminus, and amolecular weight of 20.7 kilodaltons and a deduced isoelectric point of6.27. Advantageously, the novel PPI according to this aspect of theinvention is obtainable from a fungal organism, such as a filamentousfungus, for example those used in the biotechnology industry; preferablyAspergillus, Trichoderma or Penicillium; preferably A. niger.

An example of a PPI according to the invention is set forth in SEQ IDNo. 2. The molecule identified in this sequence is obtainable fromAspergillus niger and is referred to herein as CYPB. PPI enzymes whichsatisfy the criteria set forth above are referred to herein in generalas “CYPB” enzymes. In a preferred aspect, therefore, the inventionprovides CYPB as set forth in SEQ ID No. 2, or a bioisostere thereof.

As used herein, the term “bioisostere” is used in accordance with itscommon usage in the art, to refer to namely a compound having a similar(but not the same) or a different structure and having the samebiological functional effect.

Advantageously, the bioisostere of the invention is obtainable from afungal organism, such as a filamentous fungus, for example those used inthe biotechnology industry; preferably Aspergillus, Trichoderma orPenicillium; preferably A. niger.

According to a further aspect of the present invention, there isprovided a nucleic acid encoding an enzyme according to the invention.In addition to being useful for the production of recombinant PPIprotein, these nucleic acids are also useful as probes, thus readilyenabling those skilled in the art to identify and/or isolate nucleicacid encoding PPIs or homologues thereof. The nucleic acid may beunlabelled or labelled with a detectable moiety. Furthermore, nucleicacid according to the invention is useful e.g. in a method determiningthe presence of PPI-specific nucleic acid, said method comprisinghybridising the DNA (or RNA) encoding (or complementary to) PPI to atest sample nucleic acid and determining the presence of the PPI. Inanother aspect, the invention provides nucleic acid sequence that iscomplementary to, or hybridises under stringent conditions to, a nucleicacid sequence encoding a PPI of a fragment thereof.

Advantageously, fragments of PPI-encoding nucleic acids are between 10and 200 nucleotides in length, preferably between 15 and 50 nucleotidesin length, and most preferably about 20 nucleotides in length.

The invention also provides a method for amplifying a nucleic acid testsample comprising priming a nucleic acid polymerase (chain) reactionwith nucleic acid (DNA or RNA) encoding (or complementary to) the PPI.

In still another aspect of the invention, the nucleic acid is DNA andfurther comprises a replicable vector comprising the nucleic acidencoding the PPI operably linked to control sequences recognised by ahost transformed by the vector. Furthermore the invention provides hostcells transformed with such a vector and a method of using a nucleicacid encoding a PPI to effect the production of PPI, comprisingexpressing PPI-encoding nucleic acid in a culture of the transformedhost cells and, if desired, recovering PPI from the host cell culture.

Furthermore, the present invention relates to isolated PPI proteins andbioisosteres thereof encoded by the above-described nucleic acids.

Isolated PPI nucleic acid includes nucleic acid that is free from atleast one contaminant nucleic acid with which it is ordinarilyassociated in the natural source of PPI nucleic acid or in crude nucleicacid preparations, such as DNA libraries and the like. Isolated nucleicacid thus is present in other than in the form or setting in which it isfound in nature. However, isolated PPI encoding nucleic acid includesPPI nucleic acid in ordinarily PPI-expressing cells where the nucleicacid is in a chromosomal location different from that of natural cellsor is otherwise flanked by a different DNA sequence than that found innature.

In accordance with the present invention, there are provided isolatednucleic acids, e.g. DNAs or RNAs, encoding CYPB having the sequence setforth in SEQ. ID. No. 2, or fragments thereof. In particular, theinvention provides a DNA molecule encoding CYPB as set forth in SEQ. ID.No. 2, or a fragment thereof. By definition, such a DNA comprises acoding single stranded DNA, a double stranded DNA of said coding DNA andcomplementary DNA thereto, or this complementary (single stranded) DNAitself.

The preferred sequence encoding CYPB is that having substantially thesame nucleotide sequence as the coding sequences in SEQ ID No. 2, withthe nucleic acid having the same sequence as the coding sequence in SEQID No. 2 being most preferred. As used herein, nucleotide sequenceswhich are substantially the same share at least about 90% identity.

The nucleic acids of the invention, whether used as probes or otherwise,are preferably substantially homologous to the sequence of CYPB as shownin SEQ ID No. 2. “Substantial homology”, where homology indicatessequence identity, means more than 40% sequence identity, preferablymore than 45% sequence identity and most preferably a sequence identityof 50% or more, as judged by direct sequence alignment and comparison.

Substantially homologous amino acid sequences and nucleotide sequencescan have greater than 75% homology (e.g., at least 80% homology, or atleast 85% homology, such as at least 90% homology, or even at least 95%homology, for instance at least 97% homology). Nucleotide sequencehomology can be determined using the “Align” program of Myers andMiller, (“Optimal Alignments in Linear Space”, CABIOS 4, 11-17, 1988,incorporated herein by reference) and available at NCBI. Alternativelyor additionally, the term “homology”, for instance, with respect to anucleotide or amino acid sequence, can indicate a quantitative measureof homology between two sequences. The percent sequence homology can becalculated as (N_(ref)−N_(dif))*100/N_(ref), wherein N_(dif) is thetotal number of non-identical residues in the two sequences when alignedand wherein N_(ref) is the number of residues in one of the sequences.Hence, the DNA sequence AGTCAGTC will have a sequence similarity of 75%with the sequence AATCAATC (N_(ref)=8; N_(dif)=2). Alternatively oradditionally, “homology” with respect to sequences can refer to thenumber of positions with identical nucleotides or amino acids divided bythe number of nucleotides or amino acids in the shorter of the twosequences wherein alignment of the two sequences can be determined inaccordance with the Wilbur and Lipman algorithm (Wilbur and Lipman, 1983PNAS USA 80:726, incorporated herein by reference), for instance, usinga window size of 20 nucleotides, a word length of 4 nucleotides, and agap penalty of 4, and computer-assisted analysis and interpretation ofthe sequence data including alignment can be conveniently performedusing commercially available programs (e.g., Intelligenetics™ Suite,Intelligenetics Inc. CA). When RNA sequences are said to be similar, orhave a degree of sequence identity or homology with DNA sequences,thymidine (T) in the DNA sequence is considered equal to uracil (U) inthe RNA sequence.

RNA sequences within the scope of the invention can be derived from DNAsequences, by thymidine (T) in the DNA sequence being considered equalto uracil (U) in RNA sequences.

Additionally or alternatively, amino acid sequence similarity oridentity or homology can be determined using the BlastP program(Altschul et al., Nucl. Acids Res. 25, 3389-3402, incorporated herein byreference) and available at NCBI, advantageously using defaultparameters. The following references (each incorporated herein byreference) provide algorithms for comparing the relative identity orhomology of amino acid residues of two proteins, and additionally oralternatively with respect to the foregoing, the teachings in thesereferences can be used for determining percent homology or identity:Needleman S B and Wunsch C D, “A general method applicable to the searchfor similarities in the amino acid sequences of two proteins,” J. Mol.Biol. 48:444-453 (1970); Smith T F and Waterman M S, “Comparison ofBio-sequences,” Advances in Applied Mathematics 2:482-489 (1981); SmithT F, Waterman M S and Sadler J R, “Statistical characterization ofnucleic acid sequence functional domains,” Nucleic Acids Res.,11:2205-2220 (1983); Feng D F and Dolittle R F, “Progressive sequencealignment as a prerequisite to correct phylogenetic trees,” J. of Molec.Evol., 25:351-360 (1987); Higgins D G and Sharp P M, “Fast and sensitivemultiple sequence alignment on a microcomputer,” CABIOS, 5: 151-153(1989); Thompson J D, Higgins D G and Gibson T J, “ClusterW: improvingthe sensitivity of progressive multiple sequence alignment throughsequence weighing, positions-specific gap penalties and weight matrixchoice, Nucleic Acid Res., 22:4673-480 (1994); and, Devereux J,Haeberlie P and Smithies O, “A comprehensive set of sequence analysisprogram for the VAX,” Nucl. Acids Res., 12: 387-395 (1984).

Preferably, nucleic acids according to the invention are fragments ofthe CYPB-encoding sequence. Fragments of the nucleic acid sequence of afew nucleotides in length, preferably 5 to 150 nucleotides in length,are especially useful as probes.

Exemplary nucleic acids can alternatively be characterised as thosenucleotide sequences which encode a CYPB protein and hybridise to theDNA sequences set forth SEQ ID No. 2, or a selected fragment of said DNAsequence. Preferred are such sequences encoding CYPB which hybridiseunder high stringency conditions to the sequence of SEQ ID No. 2 or afragment thereof as defined above.

Stringency of hybridisation refers to conditions under which polynucleicacids hybrids are stable. Such conditions are evident to those ofordinary skill in the field. As known to those of skill in the art, thestability of hybrids is reflected in the melting temperature (Tm) of thehybrid which decreases approximately 1 to 1.5° C. with every 1% decreasein sequence homology. In general, the stability of a hybrid is afunction of sodium ion concentration and temperature. Typically, thehybridisation reaction is performed under conditions of higherstringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permithybridisation of only those nucleic acid sequences that form stablehybrids in 1 M Na⁺ or an equivalent salt concentration, at 65-68° C.High stringency conditions can be provided, for example, byhybridisation in an aqueous solution containing 6×SSC, 5×Denhardt's, 1%SDS (sodium dodecyl sulphate), 0.1 Na⁺ pyrophosphate and 0.1 mg/mldenatured salmon sperm DNA as non specific competitor. Followinghybridisation, high stringency washing may be done in several steps,with a final wash (about 30 min) at the hybridisation temperature in0.2-0.1×SSC, 0.1% SDS.

Moderate stringency refers to conditions equivalent to hybridisation inthe above described solution but at about 60-62° C. In that case thefinal wash is performed at the hybridisation temperature in 1×SSC, 0.1%SDS.

Low stringency refers to conditions equivalent to hybridisation in theabove described solution at about 52-56° C. In that case, the final washis performed at the hybridisation temperature in 4×SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicatedusing a variety of buffers, e.g. formamide-based buffers, andtemperatures. Denhardt's solution and SSC are well known to those ofskill in the art as are other suitable hybridisation buffers (see, e.g.Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds.(1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.).Optimal hybridisation conditions have to be determined empirically, asthe length and the GC content of the probe also play a role.

The CYPB protein of the present invention comprises an ER retentionsignal. In a preferred aspect of the present invention, accordingly,there is provided a polypeptide possessing foldase activitycharacterised by having a capability to catalyse the cis-transisomerisation of a peptide bond on the N terminal side of prolineresidues in polypeptides, encoded by a nucleic acid capable ofhybridising under conditions of low, medium or high stringency with a 17base oligonucleotide derived from SEQ ID No. 1. Preferably, lowstringency conditions are used.

SEQ. ID. No. 1 represents a degenerated sequence encoding an ERretention signal. Since this signal is likely to be located on any CYPBprotein which is located in the ER, the presence of this sequence mayadvantageously be used to characterise and isolate CYPB polypeptides inaccordance with the present invention.

Given the guidance provided herein, the nucleic acids of the inventionare obtainable according to methods well known in the art. For example,a DNA of the invention is obtainable by chemical synthesis, usingpolymerase chain reaction (PCR) or by screening a genomic library or asuitable cDNA library prepared from a source believed to possess CYPBand to express it at a detectable level.

Chemical methods for synthesis of a nucleic acid of interest are knownin the art and include triester, phosphite, phosphoramidite andH-phosphonate methods, PCR and other autoprimer methods as well asoligonucleotide synthesis on solid supports. These methods may be usedif the entire nucleic acid sequence of the nucleic acid is known, or thesequence of the nucleic acid complementary to the coding strand isavailable. Alternatively, if the target amino acid sequence is known,one may infer potential nucleic acid sequences using known and preferredcoding residues for each amino acid residue.

An alternative means to isolate a gene encoding a PPI according to theinvention is to use PCR technology as described e.g. in section 14 ofSambrook et al., 1989. This method requires the use of oligonucleotideprobes that will hybridise to PPI nucleic acid. Strategies for selectionof oligonucleotides are described below.

Libraries are screened with probes or analytical tools designed toidentify the gene of interest or the protein encoded by it. For cDNAexpression libraries suitable means include monoclonal or polyclonalantibodies that recognise and specifically bind to CYPB;oligonucleotides of about 20 to 80 bases in length that encode known orsuspected CYPB cDNA from the same or different species; and/orcomplementary or homologous cDNAs or fragments thereof that encode thesame or a hybridising gene. Appropriate probes for screening genomic DNAlibraries include, but are not limited to oligonucleotides, cDNAs orfragments thereof that encode the same or hybridising DNA; and/orhomologous genomic DNAs or fragments thereof.

A nucleic acid encoding CYPB may be isolated by screening suitable cDNAor genomic libraries under suitable hybridisation conditions with aprobe, i.e. a nucleic acid disclosed herein including oligonucleotidesderivable from the sequences set forth in SEQ ID NO. 2. Suitablelibraries are commercially available or can be prepared e.g. from celllines, tissue samples, and the like.

As used herein, a probe is e.g. a single-stranded DNA or RNA that has asequence of nucleotides that includes between 10 and 50, preferablybetween 15 and 30 and most preferably at least about 20 contiguous basesthat are the same as (or the complement of) an equivalent or greaternumber of contiguous bases set forth in SEQ ID No. 2. The nucleic acidsequences selected as probes should be of sufficient length andsufficiently unambiguous so that false positive results are minimised.The nucleotide sequences are usually based on conserved or highlyhomologous nucleotide sequences or regions of CYPB. The nucleic acidsused as probes may be degenerate at one or more positions. The use ofdegenerate oligonucleotides may be of particular importance where alibrary is screened from a species in which preferential codon usage inthat species is not known.

Preferred regions from which to construct probes include 5′ and/or 3′coding sequences, sequences predicted to encode ligand binding sites,and the like. For example, either the full-length cDNA clone disclosedherein or fragments thereof can be used as probes. Preferably, nucleicacid probes of the invention are labelled with suitable label means forready detection upon hybridisation. For example, a suitable label meansis a radiolabel. The preferred method of labelling a DNA fragment is byincorporating α³²P dATP with the Klenow fragment of DNA polymerase in arandom priming reaction, as is well known in the art. Oligonucleotidesare usually end-labelled with γ³²P-labelled ATP and polynucleotidekinase. However, other methods (e.g. non-radioactive) may also be usedto label the fragment or oligonucleotide, including e.g. enzymelabelling, fluorescent labelling with suitable fluorophores andbiotinylation.

After screening the library, e.g. with a portion of DNA includingsubstantially the entire CYPB-encoding sequence or a suitableoligonucleotide based on a portion of said DNA, positive clones areidentified by detecting a hybridisation signal; the identified clonesare characterised by restriction enzyme mapping and/or DNA sequenceanalysis, and then examined, e.g. by comparison with the sequences setforth herein, to ascertain whether they include DNA encoding a completeCYPB (i.e., if they include translation initiation and terminationcodons). If the selected clones are incomplete, they may be used torescreen the same or a different library to obtain overlapping clones.If the library is genomic, then the overlapping clones may include exonsand introns. If the library is a cDNA library, then the overlappingclones will include an open reading frame. In both instances, completeclones may be identified by comparison with the DNAs and deduced aminoacid sequences provided herein.

In order to detect any abnormality of endogenous CYPB, genetic screeningmay be carried out using the nucleotide sequences of the invention ashybridisation probes. Also, based on the nucleic acid sequences providedherein antisense-type agents to reduce expression of CYPB, if desired,may be designed.

It is envisaged that the nucleic acid of the invention can be readilymodified by nucleotide substitution, nucleotide deletion, nucleotideinsertion or inversion of a nucleotide stretch, and any combinationthereof. Such mutants can be used e.g. to produce a CYPB mutant that hasan amino acid sequence differing from the CYPB sequences as found innature. Mutagenesis may be predetermined (site-specific) or random. Amutation which is not a silent mutation must not place sequences out ofreading frames and preferably will not create complementary regions thatcould hybridise to produce secondary mRNA structure such as loops orhairpins.

The invention is described below, for the purposes of illustration only,in the following examples:

EXAMPLE 1

Construction of an Aspergillus niger cDNA Library

A ZAP-i A. nigerN402 cDNA library is constructed from A. niger cDNA byuse of ZAP-cDNA synthesis kit from Stratagene, using the instructionsprovided by the manufacturer.

EXAMPLE 2

Screening of the Aspergillus niger cDNA Library

Approximately 2×50.000 pfu are plated on large (22×22 cm) NZY platescontaining the following medium (per liter): 5 g NaCl, 2 gMgSO_(4.)7H₂O, 5 g yeast extract, 10 g casein hydrolysate, 15 g agar, pHadjusted to 7.5 with NaOH. The medium is autoclaved, cooled to about 65°C. and poured into the plates. 240 ml of medium is used per plate.

The inoculated NZY plates are incubated overnight at 37° C. and plaquelifts of the plates are made. Two lifts are made for each plate onHybond N (Amersham) filters. The DNA is fixed using UV radiation for 4min. and the filters are hybridised as described in the following using,as the probe, a degenerate oligonucleotide that is labelled with³²P-dCTP using Terminal Transferase (Boehringer Mannheim) according thefollowing procedure. 500 pmol of the degenerate oligo nucleotide isused. After 3 min. incubation at 94° C. in a Terminal Transferasereaction buffer (Boehringer Mannheim), the mixture is chilled on ice and4 μl ³²P-dCTP is added. The labelling reaction is started by addition of10 units Terminal Transferase (Boehringer Mannheim). After incubation at37° C. for 30 min., the enzyme is heat inactivated by a 5 min.incubation at 70° C. The radio-labelled oligo nucleotide is purified ona NAP 5 column (Pharmacia—containing Sephadex G-25 medium).

The filters are prehybridised for 4 hours at 56° C. in 50 mlprehybridisation buffer containing 12.5 ml 20×SSC (0.3 M Na₃citrate, 3 MNaCl), 2.5 ml 100×Denhardt's solution, 2.5 ml 10% SDS and 32.5 ml water.300 μl 10 mg/ml denatured salmon sperm DNA is added to theprehybridisation buffer immediately before use. Followingprehybridisation, the radiolabelled oligonucleotide is added and filtersare hybridised overnight at 56° C. Next day the filters are washed twicewith 4×SSC+0.1% SDS for 30 min at 56° C.

The filters are autoradiographed for 16 hours and positive clones areisolated. A clone is counted as positive only if there is ahybridisation signal on both plaque lifts of the NZY plate.

Six putative clones are isolated and are purified by plating on smallPetri dishes, after which they are subjected to a second screening,essentially as described above. Four clones are eventually selected forthe conversion to plasmids using the Rapid Excision Kit (Stratagene).

Sequencing of the obtained plasmids is done with the Reverse andUniversal sequence primers. The plasmid containing the cypB geneencoding a cyclophilin like peptidyl prolyl cis-trans isomerase B isdesignated pPD23.

EXAMPLE 3

Characterisation of the pPD23 Plasmid Containing the cypB Gene

A restriction map of the clone is made. The fragment is cloned in theEcoRI and XhoI site of pBluescript SK+. The restriction map showing thestructure op pPD23 is shown in FIG. 1. The gene is sequenced using thecycle sequencing method. The complete sequence is shown in SEQ. ID. No.2. The sequence is determined on both strands for the whole construct.

The deduced amino acid sequence is aligned using the ClustalW programwith three cyclophilin like peptidyl prolyl cis-trans isomerases B. Thealignment is shown in FIG. 2. The above alignment shows that CYPB ishomologous to the other known CYPB sequences.

A search in the SWISS-PROT database is performed and does not show anysequences with a higher homology than those shown in the alignment (FIG.2). The sequence with the highest homology is a precursor for thecyclophilin like peptidyl prolyl cis-trans isomerase from Orpinomycessp. where the identity is found to be 63% (FIG. 3).

Residues 1-23 are recognised as a signal sequence, leaving a matureprotein of 186 amino acids with a deduced molecular weight of 20.7 kDaand a deduced iso-electric point (pI) of 6.27. The CYPB protein containsa putative E.R. retention signal at its extreme C-terminus (position209-212). One putative N-glycosylation site is found in CYPB at position139-142. A conserved motif for cyclophilin-type peptidyl-prolylcis-trans isomerase is present at position 79-96.

EXAMPLE 4

Expression of cypB in E. coli

A fragment, containing the part of the gene encoding for the mature CYPBprotein, is generated by PCR with the following primers:

upper primer:

5′ ccc ata tgg aag atg ctc agc ccc ggg gcc cca aga 3′  (SEQ. ID. No. 3)

lower primer:

5′ cga agc tta gtg gtg gtg gtg gtg gtg gct gct acc ttt ttc t 3′  (SEQ.ID. No. 4)

PCR is performed at 52° C. using pfu polymerase. The E.R. targetsequence is excluded from this construct. Furthermore, the C-terminalpart of the protein is extended with a HIS-tag. This fragment issubsequently cloned in plasmid pET-24a (+) (Novagen), allowingexpression of the gene in E.coli. This construct is transformed. toE.coli strain BL21(DE3)pLysS. Expression of cypB is induced with 1 mMIPTG which is added to the culture at OD₆₀₀=1.

E. coli expressed protein is purified by means of Immobilised MetalAffinity Chromatography (Ni-NTA resin, Qiagen) and gel filtration(Superdex 75, Pharmacia). The apparent molecular weight of the purifiedCYPB is determined on a SDS-PAGE gel to be approximately 21 kDa. Theexact molecular mass is determined by MALDI-TOF analysis, revealing amolecular weight of 21100.6 Da. N-terminal sequencing revealed the first10 amino acids of the mature sequence (EDAQPRGPK—residues 24 to 32 inSEQ.ID. No. 2).

A assay based on the stereospecific degradation of the substratesuc-Ala-Ala-Pro-Phe-pNA (Sigma) by α-chemotrypsin (Boehringer Mannheim)is set up to determine the activity of CYPB. The trans-isomer of thissubstrate is rapidly degraded by α-chemotrypsin. In water, 88% of thesubstrate is present as trans and 12% as cis-isomer. The conversion ofthe cis-isomer to the trans-isomer is rate limiting and is catalysed bypeptidyl prolyl cis-trans isomerases.

CYPB is assayed at 25° C. in 50 mM HEPES buffer pH 7.8 containing 50 μMsubstrate and 25 μM α-chemotrypsin. 0.5 nM CYPB protein is added to themixture and the absorbance at 380 nm is measured every 0.5 sec. Additionof CYPB protein clearly leads to a quicker degradation of the substrate,proving the foldase activity thereof.

EXAMPLE 5

Co-expression of CYPB and a Triacylglycerol Lipase

Lipase-expressing and CypB Plasmids

The plasmid pLIP4 (FIG. 4) comprises the entire genomic sequence of thelipA gene. This sequence is set forth in SEQ ID No: 6. CYPB is expressedfrom either ppd23d14 or ppd23d13, as shown in FIGS. 5 and 6respectively. Ppd23d14 comprises the CypB sequence under the control ofthe glaA promoter (generally available; see, for example, Ward et al.,(1995) Biotechnology 13:498-503), which is inducible. Ppd23d13 comprisesthe CypB sequence under the control of the A. nidulans gpdA promoter(Punt et al., (1991) J. Biotechnol. 17:19-34), which is constitutivelyactive. In both cases, the CypB gene is followed by the A. nidulans trpCterminator.

Antibiotic resistance is incorporated into the plasmids by insertion ofa hygromycin resistance gene (isolated from Streptomyces hygroscopicusand E. coli) under the control of the gpdA promoter.

Transformation of the Aspergillus tubigensis Strain 3M pyrA with theLipase Gene

Spores from the A. tubigensis strain 3M pyr A are cultivated overnightat 34° C. in a shake flask containing minimal medium supplemented with2% glucose and 10 mM uridine. The mycelium is harvested and resuspendedin lysis buffer plus lysing enzyme. The protoplasts produced are mixedwith pLIP4, together with ppd23d14 or ppd23d13, by cotransformation,using pyrG and antibiotic resistance markers to select for the desiredrecombinants.

In Situ Detection of Lipase Production in Transformed AspergillusStrains

A screening procedure used to visualise fungal lipase after ultrathinlayer isoelectric focusing is adapted to screen Aspergillustransformants grown on agar plates. This procedure is very convenientfor the initial analysis of expressing and non-expressing transformedAspergillus strains. Screening of lipase producers on agar plates isdone using 2% olive oil as the substrate of the enzyme (lipase) as wellas the inducer of the lipase promoter. In addition, the plate contains afluorescent dye Rhodamine B(N-9-(2-carboxyphenyl)-6-(diethylamino)-3H-xanthen-3-ylidene-N-ethylethanaminiumchloride). In the presence of olive oil, the transformants are inducedto secrete lipase. The lipase secreted into the agar plate hydrolysesthe olive oil causing the formation of orange fluorescent colonies thatare visible upon UV irradiation (350 nm). The detection of fluorescentcolonies is observed after about 24 hours of growth, depending on thetransformant. After several days of growth, the lipase producing strainscan be identified as orange fluorescent strains that are visible by eye.Under these plate screening conditions, the untransformed strain give nobackground fluorescence and appear as opaque pink colonies. However, oneshould be conscious of possible contaminating yeast and bacterialstrains that can grow rapidly on the oil containing plates.Contamination is prevented by the incorporation ofantibiotics—Ampicillin.

Characterisation of Lipase Secreting Transformants

The 16 transformants that show orange fluorescent halos are cultivatedin shake flasks containing 100 ml of minimal medium plus 1% olive oil,0.5% yeast extract, 0.2% casamino acids and grown for 8 days. The amountof lipase secreted is quantitated by applying 10 μl of cell-free culturesupernatant into holes punched in the olive oil-rhodamine B agar platesand incubating the plates overnight at 37° C. Using this technique, thecell free culture supernatant from the 5 transformants that give themost intense fluorescence are further analysed by chromatography.

Purification of Recombinant Lipase by Hydrophobic InteractionChromatography (HIC)

Culture supernatant from the five different lipase secretingtransformants found positive by the plate screening method are desaltedusing NAP 5 columns (Pharmacia: contain Sephadex G-25 medium) andequilibrated in 1M (NH₄)₂SO₄ 50 mM sodium acetate pH 5.5. The desaltedculture supernatant is fractionated by hydrophobic interactionchromatography on a Biogel Phenyl-5 PW Column (Biorad). Elution is doneby a descending salt gradient of 1M to zero Molar (NH₄)₂SO₄, 20 mMsodium acetate, pH 5.5. A single discrete protein peak is observed afterfractionation. The area of the protein peaks is calculated among thedifferent transformants and compared with the untransformed strains. Thetable below summarises the levels of Lipase secreted by the 5transformants. The best transformant shows a 62 fold increased in theamount of lipase purified after HIC fractionation. The table also showsthe varying amounts of lipase produced by the different transformantsafter 6 days of growth under unoptimised small scale shake flaskcondition.

Levels of Secreted Lipase after HIC = Area of the discrete proteinpeak/area of 6M) Transformants grown for 6 days in Area = height × FWHM(full width 1% olive oil as the carbon source half median) L43-6 Flipper61.9 L3-6 10.5 L1-6 13.1 L13-6 17.0 L47-6 29.3 6M-6 Untransformed 6Mstrain  1.0

Characterisation of Recombinant Lipase

1. Amino Acid Analysis and Protein Determination

The discrete protein peak after fractionation by HIC is freeze dried andresuspended in water. The amino acid composition and the proteinconcentration of the purified lipase protein are determined to obtain acorrelation coefficient between UV absorbance at 280 nm and proteinconcentration. This allows the estimation of Lipase concentration inhomogenous preparations.

The Lipase protein is carboxymethylated and the sequence of the first 15amino acids is determined by N-terminal amino acid sequencing. The 15amino acid sequence of the recombinant lipase is exactly the same as thenative lipase indicating correct signal sequence cleavage.

2. SDS-PAGE Electrophoresis

The different protein factions collected after HIC are separated on a12% Tris-Glycine SDS gel. Silver staining reveals one protein band,confirming the homogeneity of the protein peaks. In addition, the crudeextract shows a major lipase band as the only protein band thataccumulated in the culture supernatant in very high amounts when thefungus is cultured in medium containing oil.

3. Detection of the Presence of a Covalently Attached N-linkedOligosaccharides in Recombinant Lipase

The detection of N-linked oligosaccharides is achieved by digestion ofthe lipase with Endo- -N-acetyl-glucosamidase H from Streptomyces(Sigma). Endo H treatment of recombinant lipase secreted into the growthmedium alters the mobility of the band seen on SDS-PAGE and runs as asingle species with a molecular mass of approximately 30 kDa. Thisindicates the extent of N-linked glycosylation.

4. Matrix Assisted Laser Desorption Mass Spectrometry Recombinant Lipase

MALDI-TOF mass spectrometry is performed using purified lipase mixedwith a matrix solution consisting of sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid) in 70% acetonitrile, 0.1% TFA.The molecular mass determined from the desalted recombinant lipase is32,237 Daltons.

Deglycosylated lipase generated by digestion with endoglycosidase H andanalysed directly by Maldi-MS gave an estimate of the molecular weightof the polypeptide backbone of 29.325 Da.

Using this analysis, the presence and the approximate number of N-linkedoligosaccharides on the glycoprotein can be determined. In conclusion,N-linked oligosaccharides account for approximately 10% of the molecularweight of recombinant lipase.

Mass Spectrometry analysis of LIPASE + endoglycosidase H recombinantlipase 32,237 daltons 29,326 daltons native lipase 30,310 daltons 29,333daltons

EXAMPLE 6

Overexpression of cypB in Aspergillus niger

Aspergillus strain N592 (cspA1, pyrA5) has been transformed with aplasmid allowing expression of cypB under the strong constitutive gpdApromoter. Putative transformants are screened by PCR to confirm thepresence of this plasmid. The number of integrated copies and the levelof cypB expression in this strain (N592::pPD23d13) is determined bySouthern and Northern analysis respectively.

The Southern analysis shows clearly that strain N592::pPD23d13 containsmultiple copies of the integrated plasmid pPD23d13. The level ofexpression of cypB in this strain is approximately 15 times higher thanthe wild type expression level.

To determine whether this strain is capable of secreting more protein, agrowth experiment is set up in which the wild type strain (N402) andN592::pPD23d13 are grown in the presence of 2% (w/v) starch. Starch isknown to be a specific inducer for the expression of glucoamylase.Supernatant samples were therefor assayed for glucoamylase activity andamounts of secreted proteins.

Glucoamylase is regarded as a well secreted protein and it is commonlyused as a fusion protein to aid the secretion of difficult targetproteins.

The overexpression of cypB clearly leads to an increased productionlevel of glucoamylase. An almost two fold increase of glucoamylaseactivity is measured after 72 hours of induction. No de novoglucoamylase production is seen at this time, most likely because of adepletion of the inducing carbon source.

EXAMPLE 7

Localisation of CYPB

Secretion of proteins from eukaryotic cells is a complex process. Newlysynthesised secretory and membrane proteins enter the endoplasmicreticulum (ER) in an unfolded state and must acquire a specificconformation before they can be transported further within the secretorypathway. A number of proteins have been found within the lumen of theER. These include BiP (binding protein, a homologue of the 70 kDaheat-shock protein), protein disulphide isomerase (PDI) and peptidylprolyl cis-trans isomerase (PPI). These foldases are involved incatalysing the folding of a protein from the unfolded to the nativestate. In order to remain in the ER, and therefor to be diverted fromthe bulk flow of secreted proteins, foldases have specific retention andretrieval signal. A common carboxy terminal tetrapeptide, HDEL has beenidentified as a signal capable of retaining a protein within the lumenof the ER. Removal of this tetrapeptide leads to secretion of theprotein (Pelham (1990) Trends Biochem. Sci. 15, 483-486)

The CYPB protein contains a signal sequence and a putative ER retentionsignal, indicating that the protein is targeted and retained in the ER.However the retention signal in this protein is slightly divergent fromthe known retention signals. At position −3 (counting from the lastamino acid residue) the CYPB protein contains a glutamic acid residue.This HEEL sequence has not been identified as an ER retention signal. Toevaluate if this sequence is capable of retaining CYPB within the lumenof the ER, a GFP construct is made containing both the CYPB signalsequence and the CYPB ER retention signal. Expression of this gene isdriven by the strong, constitutive gpdA promoter (plasmid pPD38d3; FIG.7).

Transformants of the Aspergillus niger strain D15 (prtT; pyrG; phmA) arescreened by PCR for insertion of the GFP expression plasmid. Strainscontaining the expression constructs are analysed for the expression ofGFP.

Strain D15::pPD38d3#5 is grown overnight in liquid cultures and showedthat GFP is directed to a tubular network within the cell. Equivalentconstructs in A. nidulans have been demonstrated to target GFP to theER, illuminating a network similar, to our findings (Fernandez-Abalos,et al. (1998) Mol. Microbiol. 27, 121-130). Staining of hyphae withER-Tracker DPX (Molecular Probes) illuminates the same type of tubularnetwork.

Finally DIOC₆, a stain for mitochondria at low concentration, but alsoeffective for ER staining when applied at higher concentrations, revealsthe same tubular network as seen for DPX and GFP. DIOC₆ staining ishowever hampered by a diffusion of the stain out of the ER, resultingonly in a staining of the mitochondria.

Our results show clearly that HEEL (SEQ ID NO: 8) is both necessary andsufficient for GFP retention in the ER. It is therefore clear that theCYPB protein is targeted to and retained in the ER.

12 1 17 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 1 ggyyavagyt crtcrtg 17 2 987 DNA Aspergillusniger CDS (42)..(677) 2 ggcacgagaa ttctcctaca ttggagacat cctgagcaat catg aac ttc aag aac 56 Met Asn Phe Lys Asn 1 5 att ttt cta tct ttc ttcttc gtc ctg gcg gtt gga ctt gct ctt gtc 104 Ile Phe Leu Ser Phe Phe PheVal Leu Ala Val Gly Leu Ala Leu Val 10 15 20 cac gcc gaa gat gct cag ccccgg ggc ccc aag atc acc agt aag gtg 152 His Ala Glu Asp Ala Gln Pro ArgGly Pro Lys Ile Thr Ser Lys Val 25 30 35 ttc ttt gat ata gag cac gga gacaag cct ctg ggc aga gtc gtg ctt 200 Phe Phe Asp Ile Glu His Gly Asp LysPro Leu Gly Arg Val Val Leu 40 45 50 ggc ttg tat ggc aag act gtt cct aagacc gct gag aac ttc cgg gct 248 Gly Leu Tyr Gly Lys Thr Val Pro Lys ThrAla Glu Asn Phe Arg Ala 55 60 65 ctc gct act ggt gag aag ggc ttt ggc tatgaa gga tct acc ttc cac 296 Leu Ala Thr Gly Glu Lys Gly Phe Gly Tyr GluGly Ser Thr Phe His 70 75 80 85 cgt gtc att aag gac ttc atg atc cag ggtggt gac ttc act cgt ggc 344 Arg Val Ile Lys Asp Phe Met Ile Gln Gly GlyAsp Phe Thr Arg Gly 90 95 100 gat ggt acc ggt gga aag tcg atc tac ggtgag aag ttc gcc gac gaa 392 Asp Gly Thr Gly Gly Lys Ser Ile Tyr Gly GluLys Phe Ala Asp Glu 105 110 115 aac ttc aag ctg agg cat acg cgc aag gggctc ctg agc atg gcc aac 440 Asn Phe Lys Leu Arg His Thr Arg Lys Gly LeuLeu Ser Met Ala Asn 120 125 130 gcc ggc aag gac acc aac ggc tcc cag ttcttc atc acc acc gtt cct 488 Ala Gly Lys Asp Thr Asn Gly Ser Gln Phe PheIle Thr Thr Val Pro 135 140 145 aca cct tgg ctt gat ggc cgc cat gtc gtcttc ggt gaa gtg ctc gag 536 Thr Pro Trp Leu Asp Gly Arg His Val Val PheGly Glu Val Leu Glu 150 155 160 165 ggc tac gag atc gtc gct cag att gagaac gtg ccc aag ggc cgt tct 584 Gly Tyr Glu Ile Val Ala Gln Ile Glu AsnVal Pro Lys Gly Arg Ser 170 175 180 gac aga ccc gtg gag act gtc aag atcgtc aag agt gga gag ttg gag 632 Asp Arg Pro Val Glu Thr Val Lys Ile ValLys Ser Gly Glu Leu Glu 185 190 195 tct gag gac aag gct gga gaa aaa ggtagc agc cac gag gag ctg 677 Ser Glu Asp Lys Ala Gly Glu Lys Gly Ser SerHis Glu Glu Leu 200 205 210 tagacctgtt tcctgaggtc tcggccytgc ttctcgataaractgtgaat gtgcydaacc 737 gcttkgtaaa gaaacgagct ccgaagaaga gtcacaaccttcagcaattg ctgttattcc 797 ttctccaacc cctttgccta tgacatctga taacgcyccttatattttcc cgaaattcgc 857 agcgcttgcc attgttgtcg gtcycctggt tgtcctggtccgacgcagct caagggagaa 917 caagagaagg ctgaaagaag tattgtttga tagaaattgtactcatccaa wtaaaaaaaa 977 aaaaaaaaaa 987 3 36 DNA Artificial SequenceDescription of Artificial Sequence Primer 3 cccatatgga agatgctcagccccggggcc ccaaga 36 4 43 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 cgaagcttag tggtggtggt ggtggtggct gctacctttttct 43 5 212 PRT Aspergillus niger 5 Met Asn Phe Lys Asn Ile Phe Leu SerPhe Phe Phe Val Leu Ala Val 1 5 10 15 Gly Leu Ala Leu Val His Ala GluAsp Ala Gln Pro Arg Gly Pro Lys 20 25 30 Ile Thr Ser Lys Val Phe Phe AspIle Glu His Gly Asp Lys Pro Leu 35 40 45 Gly Arg Val Val Leu Gly Leu TyrGly Lys Thr Val Pro Lys Thr Ala 50 55 60 Glu Asn Phe Arg Ala Leu Ala ThrGly Glu Lys Gly Phe Gly Tyr Glu 65 70 75 80 Gly Ser Thr Phe His Arg ValIle Lys Asp Phe Met Ile Gln Gly Gly 85 90 95 Asp Phe Thr Arg Gly Asp GlyThr Gly Gly Lys Ser Ile Tyr Gly Glu 100 105 110 Lys Phe Ala Asp Glu AsnPhe Lys Leu Arg His Thr Arg Lys Gly Leu 115 120 125 Leu Ser Met Ala AsnAla Gly Lys Asp Thr Asn Gly Ser Gln Phe Phe 130 135 140 Ile Thr Thr ValPro Thr Pro Trp Leu Asp Gly Arg His Val Val Phe 145 150 155 160 Gly GluVal Leu Glu Gly Tyr Glu Ile Val Ala Gln Ile Glu Asn Val 165 170 175 ProLys Gly Arg Ser Asp Arg Pro Val Glu Thr Val Lys Ile Val Lys 180 185 190Ser Gly Glu Leu Glu Ser Glu Asp Lys Ala Gly Glu Lys Gly Ser Ser 195 200205 His Glu Glu Leu 210 6 1834 DNA Aspergillus niger modified_base (3)a, t, c, g, other or unknown 6 ccndttaatc ccccaccggg gttcccgctcccggatggag atggggccaa aactggcaac 60 ccccagttgc gcaacggaac aaccgccgacccggaacaaa ggatgcggat gaggagatac 120 ggtgcctgat tgcatggctg gcttcatctgctatcgtgac agtgctcttt gggtgaatat 180 tgttgtctga cttaccccgc ttcttgctttttcccccctg aggccctgat ggggaatcgc 240 ggtgggtaat atgatatggg tataaaagggagatcggagg tgcagttgga ttgaggcagt 300 gtgtgtgtgt gcattgcaga agcccgttggtcgcaaggtt ttggtcgcct cgattgtttg 360 tataccgcaa gatgttctct ggacggtttggagtgctttt gacagcgctt gctgcgctgg 420 gtgctgccgc gccggcaccg cttgctgtgcggagtaggtg tgcccgatgt gagatggttg 480 gatagcactg atgaagggtg aataggtgtctcgacttcca cgttggatga gttgcaattg 540 ttcgcgcaat ggtctgccgc agcttattgctcgaataata tcgactcgaa agactccaac 600 ttgacatgca cggccaacgc ctgtccatcagtcgaggagg ccagtaccac gatgctgctg 660 gagttcgacc tgtatgtcac tcagatcgcagacatagagc acagctaatt tgaacaggac 720 gaacgacttt ggaggcacag ccggtttcctggccgcggac aacaccaaca agcggctcgt 780 ggtcgccttc cggggaagca gcacgattgagaactggatt gctaatcttg acttcatcct 840 ggaagataac gacgacctct gcaccggctgcaaggtccat actggtttct ggaaggcatg 900 ggagtccgct gccgacgaac tgacgagcaagatcaagtct gcgatgagca cgtattcggg 960 ctatacccta tacttcaccg ggcacagtttgggcggcgca ttggctacgc tgggagcgac 1020 agttctgcga aatgacggat atagcgttgagctggtgagt ccttcacaaa ggtgatggag 1080 cgacaatcgg gttctgacag tcaatagtacacctatggat gtcctcgaat cggaaactat 1140 gcgctggctg agcatatcac cagtcagggatctggggcca acttccgtgt tacacacttg 1200 aacgacatcg tcccccgggt gccacccatggactttggat tcagtcagcc aagtccggaa 1260 tactggatca ccagtggcaa tggagccagtgtcacggcgt cggatatcga agtcatcgag 1320 ggaatcaatt caacggcggg aaatgcaggcgaagcaacgg tgagcgttgt ggctcacttg 1380 tggtactttt ttgcgatttc cgagtgcctgctataactag accgactgtc agattagtgg 1440 acgggagaag tgtacataag taattagtatataatcagag caacccagtg gtggtgatgg 1500 tggtgaaaga agaaacacat tgagttcccattacgkagca gwtaaagcac ktkkggaggc 1560 gctggttcct ccacttggca gttggcggccatcaatcatc tttcctctcc ttactttcgt 1620 ccaccacaac tcccatcctg ccagctgtcgcatccccggg ttgcaacaac tatcgcctcc 1680 ggggcctccg tggttctcct atattattccatccgacggc cgacgtttca ccctcaacct 1740 gcgccgccgc aaaatctccc cgagtcggtcaactccctcg aaccgccgcc cgcatcgacc 1800 tcaccgaccc cgaccgtctg ygatygtccaaccg 1834 7 4 PRT Artificial Sequence Description of Artificial SequenceIllustrative ER retention signal 7 His Asp Glu Leu 1 8 4 PRT ArtificialSequence Description of Artificial Sequence Illustrative ER retentionsignal 8 His Glu Glu Leu 1 9 4 PRT Artificial Sequence Description ofArtificial Sequence Illustrative ER retention signal 9 Lys Asp Glu Leu 110 203 PRT Orpinomyces sp. 10 Met Asn Phe Ser Ile Lys Ser Val Ile PheLeu Ala Ile Val Ala Leu 1 5 10 15 Ala Thr Leu Val Ser Ala Ser Thr AsnPro Lys Val Thr Asn Lys Val 20 25 30 Tyr Phe Asp Ile Lys Gln Gly Asp LysAsp Leu Gly Arg Ile Val Leu 35 40 45 Gly Leu Tyr Gly Glu Val Val Pro LysThr Val Glu Asn Phe Arg Ala 50 55 60 Leu Ala Thr Gly Glu Lys Gly Tyr GlyTyr Lys Asn Ser Lys Phe His 65 70 75 80 Arg Val Ile Lys Asp Phe Met IleGln Gly Gly Asp Phe Thr Arg Gly 85 90 95 Asp Gly Thr Gly Gly Lys Ser IleTyr Gly Glu Arg Phe Ala Asp Glu 100 105 110 Asn Phe Lys Leu Arg His ThrGly Pro Gly Ile Leu Ser Met Ala Asn 115 120 125 Ala Gly Arg Asp Thr AsnGly Ser Gln Phe Phe Ile Thr Thr Val Thr 130 135 140 Thr Ser Trp Leu AspGly Arg His Val Val Phe Gly Lys Val Ile Glu 145 150 155 160 Gly Met AspVal Val Thr Ala Ile Glu Thr Thr Lys Thr Leu Pro Gly 165 170 175 Asp ArgPro Ala Thr Pro Val Ile Ile Ala Asp Cys Gly Glu Leu Pro 180 185 190 ValSer Asn Asn Asn Asp Ala Lys Ala Glu Leu 195 200 11 207 PRT Mus musculus11 Met Lys Ala Leu Val Ala Ala Thr Ala Leu Gly Pro Ala Leu Leu Leu 1 510 15 Leu Leu Pro Ala Ala Ser Arg Ala Asp Glu Arg Lys Lys Gly Pro Lys 2025 30 Val Thr Ala Lys Val Phe Phe Asp Leu Arg Val Gly Glu Glu Asp Ala 3540 45 Gly Arg Val Val Ile Gly Leu Phe Gly Lys Thr Val Pro Lys Thr Val 5055 60 Glu Asn Phe Val Ala Leu Ala Thr Gly Glu Lys Gly Phe Gly Phe Lys 6570 75 80 Gly Ser Lys Phe His Arg Val Ile Lys Asp Phe Met Ile Gln Gly Gly85 90 95 Asp Phe Thr Arg Gly Asp Gly Thr Gly Gly Lys Ser Ile Tyr Gly Asp100 105 110 Arg Phe Pro Asp Glu Asn Phe Lys Leu Lys His Tyr Gly Pro GlyTrp 115 120 125 Val Ser Met Ala Asn Ala Gly Lys Asp Thr Asn Gly Ser GlnPhe Phe 130 135 140 Ile Thr Thr Val Lys Thr Ala Trp Leu Asp Gly Lys HisVal Val Phe 145 150 155 160 Gly Lys Val Leu Glu Gly Met Asp Val Val ArgLys Val Glu Asn Thr 165 170 175 Lys Thr Asp Ser Arg Asp Lys Pro Leu LysAsp Val Thr Ile Ala Asp 180 185 190 Cys Gly Thr Ile Glu Val Glu Lys ProPhe Ala Ile Ala Lys Glu 195 200 205 12 208 PRT Homo sapiens 12 Met LysVal Leu Leu Ala Ala Ala Leu Ile Ala Gly Ser Val Phe Phe 1 5 10 15 LeuLeu Leu Pro Gly Pro Ser Ala Ala Asp Glu Lys Lys Lys Gly Pro 20 25 30 LysVal Thr Val Lys Val Tyr Phe Asp Leu Arg Ile Gly Asp Glu Asp 35 40 45 ValGly Arg Val Ile Phe Gly Leu Phe Gly Lys Thr Val Pro Lys Thr 50 55 60 ValAsp Asn Phe Val Ala Leu Ala Thr Gly Glu Lys Gly Phe Gly Tyr 65 70 75 80Lys Asn Ser Lys Phe His Arg Val Ile Lys Asp Phe Met Ile Gln Gly 85 90 95Gly Asp Phe Thr Arg Gly Asp Gly Thr Gly Gly Lys Ser Ile Tyr Gly 100 105110 Glu Arg Phe Pro Asp Glu Asn Phe Lys Leu Lys His Tyr Gly Pro Gly 115120 125 Trp Val Ser Met Ala Asn Ala Gly Lys Asp Thr Asn Gly Ser Gln Phe130 135 140 Phe Ile Thr Thr Val Lys Thr Ala Trp Leu Asp Gly Lys His ValVal 145 150 155 160 Phe Gly Lys Val Leu Glu Gly Met Glu Val Val Arg LysVal Glu Ser 165 170 175 Thr Lys Thr Asp Ser Arg Asp Lys Pro Leu Lys AspVal Ile Ile Ala 180 185 190 Asp Cys Gly Lys Ile Glu Val Glu Lys Pro PheAla Ile Ala Lys Glu 195 200 205

What is claimed is:
 1. A method for producing a secretable polypeptidein a host cell, comprising over-expressing a peptidyl prolyl cis-transisomerase in the host cell, thereby increasing the yield of the secretedpolypeptide, wherein the peptidyl prolyl cis-trans isomerase comprises asignal sequence at its N-terminus and an endoplasmic reticulum retentionsignal at its C-terminus.
 2. A method according to claim 1, comprisingcotransfecting the cell with a first coding sequence encoding thepolypeptide and a second coding sequence encoding a peptidyl prolylcis-trans isomerase.
 3. A method according to claim 2, wherein thepolypeptide and the peptidyl prolyl cis-trans isomerase are encoded onseparate vectors.
 4. A method according to claim 1, wherein the codingsequence encoding that peptidyl prolyl cis-trans isomerase is integratedinto the genome of the host cell.
 5. A method according to claim 1,wherein the cell is a fungal cell.
 6. A method according to claim 1,wherein the peptidyl prolyl cis-trans isomerase is of fungal origin. 7.A method according to claim 1, wherein the endoplasmic reticulumretention signal is H D E L (SEQ ID NO: 7), H E E L (SEQ ID NO: 8) or KD E L (SEQ ID NO: 9).
 8. A method for producing a secretable polypeptidein a host cell, comprising overexpressing a peptidyl prolyl cis-transisomerase in the host cell, thereby increasing the yield of the secretedpolypeptide, wherein the peptidyl prolyl cis-trans isomerase is apolypeptide: (1) possessing foldase activity, (2) having a capability tocatalyze the cis-trans isomerisation of a peptide bond on the N terminalside of proline residues in polypeptides, (3) having a signal sequenceat the N-terminus, (4) having an endoplasmic reticulum retention signalat the C-terminus, (5) having a molecular weight of 20.7 kDa, and (6)having a deduced isoelectric point of 6.27.
 9. A nucleic acid vectorencoding a polypeptide comprising the amino acid sequence shown in SEQID NO: 5 or an amino acid sequence having at least 75% homology with SEQID NO:
 5. 10. A host cell transformed with a vector according to claim9.
 11. A host cell according to claim 10, which is a fungal host cell.12. A host cell according to claim 11, which is an Aspergillus hostcell.
 13. A process for producing a polypeptide possessing foldaseactivity characterized by having a capability to catalyze the cis-transisomerisation of a peptide bond on the N-terminal side of prolineresidues in polypeptides, comprising transforming a host cell with avector according to claim
 9. 14. A method for producing a secretablepolypeptide in a host cell, comprising overexpressing a peptidyl prolylcis-trans isomerase in the host cell, thereby increasing the yield ofthe secreted polypeptide, wherein the peptidyl prolyl cis-transisomerase is a polypeptide possessing foldase activity having acapability to catalyse the cis-trans isomerisation of a peptide bond onthe N terminal side of proline residues in polypeptides, said peptidylprolyl cis-trans isomerase being encoded by a nucleic acid capable ofhybridising under high stringency conditions with a 20 baseoligonucleotide derived from SEQ ID No:
 2. 15. A method for producing asecretable polypeptide in a host cell, comprising overexpressing apeptidyl prolyl cis-trans isomerase in the host cell, thereby increasingthe yield of the secreted polypeptide, wherein the peptidyl prolylcis-trans isomerase is a polypeptide possessing foldase activity havinga capability to catalyse the cis-trans isomerisation of a peptide bondon the N terminal side of proline residues in polypeptides, saidpeptidyl prolyl cis-trans isomerase being encoded by a nucleic acidcapable of hybridising under high stringency conditions with a 17 baseoligonucleotide derived from SEQ ID No:
 1. 16. A method for producing asecretable polypeptide in a host cell, comprising overexpressing apeptidyl prolyl cis-trans isomerase in the host cell, thereby increasingthe yield of the secreted polypeptide, wherein the peptidyl prolylcis-trans isomerase is a polypeptide possessing foldase activity havinga capability to catalyse the cis-trans isomerisation of a peptide bondon the N terminal side of proline residues in polypeptides, saidpeptidyl prolyl cis-trans isomerase being encoded by a nucleic acidwhich is at least 80% homologous to SEQ ID No:
 2. 17. A method forproducing a secretable polypeptide in a host cell, comprisingoverexpressing a peptidyl prolyl cis-trans isomerase in the host cell,thereby increasing the yield of the secreted polypeptide, wherein thepeptidyl prolyl cis-trans isomerase is a polypeptide comprising theamino acid sequence shown in SEQ ID No: 5 or an amino acid sequencehaving at least 75% homology with SEQ ID No:
 5. 18. A nucleic acidvector comprising a nucleic acid comprising the nucleotide sequenceshown as SEQ ID No: 2 or a nucleotide sequence having at least 80%homology to SEQ ID No: 2.